Polyketide synthases (PKSs) use a multi-step process to generate structurally complex natural products with wide ranging pharmaceutical and agricultural applications. Over the past decade there has been significant interest and notable successes in the generation of hybrid PKSs. These metabolic systems are attractive due to their potential to generate new natural products with valuable applications including drug discovery and development. Nonetheless, a significant and unresolved problem in this endeavor has been the significant finding that hybrid modular PKS systems are either catalytically inefficient, or simply inactive. The ongoing discovery of natural product biosynthetic gene clusters has recently provided new sets of genetic tools for the generation of more efficient hybrid PKSs. Recent advances in methods for genetic manipulation of large biosynthetic gene clusters now permit rapid generation of libraries of hybrid systems, and bioactivity assays can be used to identify those with significant catalytic activity. We have also developed a new chemoenzymatic approach for generating novel polyketide structures that can utilize both native and hybrid PKSs. This project will capitalize on these recent advances and demonstrate how they can be applied and combined to generate new biologically active macrolide antibiotics. The work will be based on the pikromycin PKS (Pik PKS)that generates a ketolide macrolactone core molecule (a class of antibiotics useful for treatment of multidrug resistant pathogens). The four specific aims are as follows: 1) chemoenzymatic synthesis of new macrolide products; 2) relaxing the specificity, and evolving iterative properties of ketoacyl acyl carrier protein synthase domains; 3) developing new methods for efficient initiation utilizing different substrates; and 4) efficiently and selectively altering extender unit specificity. Overall this project will provide new techniques and genetic tools that can be applied to other PKSs systems, including catalytically impaired hybrid PKSs, and will represent an important step towards the ultimate goal of using of combinatorial biosynthesis and chemoenzymatic synthesis for practical drug development.